Immunomodulatory methods for treatment of atherosclerosis via inhibition of CD4+ T cell response to APOB100

ABSTRACT

Immunostimulatory methods and systems for treating or preventing atherosclerosis and/or a condition associated thereto in an individual.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to the Swedish patent application No.0950161-0, filed on Mar. 17, 2009 entitled “Abrogation of T cellResponse to Low Density Lipoprotein as a Treatment for Atherosclerosis”,which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to immunomodulatory methods and systemsthat are particularly suitable for treatment and/or prevention ofatherosclerosis and/or conditions associated thereto and relatedproteins, peptides and compositions.

BACKGROUND

Atherosclerosis is currently viewed as a chronic lipid-related andimmunemediated inflammatory disease of the arterial walls. Many immunecomponents have been identified that participate in atherogenesis andpre-clinical studies have yielded promising results suggesting thatimmune-modulatory therapies targeting these components can reduceatherosclerosis.

SUMMARY

Provided herein, are methods and systems for inducing immunomodulatoryresponses in an individual. In several embodiments, the immunomodulatoryresponses induced by the methods and systems of the present disclosureare associated to a therapeutic or preventive effect related toatherosclerosis in the individual or a condition associated thereto.

According to a first aspect, a method and system to treat and/or preventatherosclerosis in an individual is described. The method comprises:inhibiting in the individual a CD4⁺ T cell response to ApoB100, inparticular by administering a therapeutically effective amount of acompound capable of inhibiting said response. The system comprises oneor more agents suitable to inhibit CD4⁺ T cell response to ApoB100 ofthe individual and one or more agents suitable to detect the reducedresponse in the individual.

According to a second aspect, a method and system to treat and/orprevent atherosclerosis in an individual is described. The methodcomprises: inhibiting in the individual T cell receptor beta variable 31(TCR TRBV31), in particular by administering a therapeutically effectiveamount of a compound capable of inhibiting said receptor. The systemcomprises one or more agents suitable to inhibit T cell receptor betavariable 31 of the individual and one or more agents suitable to detectthe inhibition in the individual. Alternatively, the method comprises:inhibiting in individual a T cell receptor with a DNA sequence highlyhomologous to that of TRBV31, in particular by administering atherapeutically effective amount of a compound capable of inhibitingsaid receptor. The system comprises one or more agents suitable toinhibit such T cell receptor of the individual and one or more agentssuitable to detect the inhibition in the individual.

According to a third aspect, a method and system to treat and/or preventatherosclerosis in an individual is described. The method comprises:immunizing the individual against T cell receptor beta variable 31, forexample by administering TCR TRBV31 peptide SEQ ID NO: 1 or other TCRTRBV31 immunogenic fragments in particular from CDR2 variable region.The system comprises one or more agents suitable to immunize theindividual against T cell receptor beta variable 31 of the individualand one or more agents suitable to detect the immunization in theindividual. Alternatively, the method comprises: immunizing theindividual against a T cell receptor highly homologous to TRBV31, forexample by administering the homologous (human) TCR TRBV30 or a TCRTRBV30 immunogenic fragment in particular from CDR2 variable region. Thesystem then comprises one or more agents suitable to immunize theindividual against such a T cell receptor of the individual and one ormore agents suitable to detect the immunization in the individual.

According to a fourth aspect, a T cell receptor beta variable 31 or animmunogenic fragment thereof is described, the T cell receptor betavariable 31, for use as a medicament.

According to a fifth aspect, T cell receptor beta variable 31 or animmunogenic fragment thereof is described, the T cell receptor betavariable 31, for use in the treatment of atherosclerosis.

According to a sixth aspect, a T cell receptor highly homologous to thatof TRBV31, such as the homologous human TCR TRBV30 or a TCR TRBV30immunogenic fragment in particular from CDR2 variable region, is used inthe treatment of atheroscleros.

According to a seventh aspect, an antibody reactive to the T cellreceptor beta variable 31 (TCR TRBV31) or an immunogenic fragmentthereof for use as a medicament.

According to an eighth aspect, an antibody reactive to a T cell receptorhighly homologous to that of TRBV31, such as the homologous human TCRTRBV30 or a TCR TRBV30 immunogenic fragment in particular from CDR2variable region for use as a medicament.

According to a ninth aspect, an antibody reactive to the T cell receptorbeta variable 31 (TCR TRBV31) or an immunogenic fragment thereof for usein the treatment of atherosclerosis.

According to a tenth eleventh aspect, an antibody reactive to a T cellreceptor highly homologous to that of TRBV31, such as the homologoushuman TCR TRBV30 or a TCR TRBV30 immunogenic fragment in particular fromCDR2 variable region for use in the treatment of atherosclerosis.

According to a eleventh aspect, a composition and in particular, avaccine is described, the composition comprising at least one of the Tcell receptor beta variable 31 (TCR TRBV31), an immunogenic fragmentthereof or an antibody together with an adjuvant and/or excipient. Inseveral embodiments the adjuvant and/or excipients are pharmaceuticallyacceptable and the composition is pharmaceutical composition.

According to a twelfth aspect, a composition and in particular, avaccine is described, the composition comprising at least one of the Tcell receptor beta variable 31 (TCR TRBV31), an immunogenic fragmentthereof or an antibody together with an adjuvant and/or excipient. Inseveral embodiments the adjuvant and/or excipients are pharmaceuticallyacceptable and the composition is pharmaceutical composition.

According to a thirteenth aspect, a hybridoma from mice immunized withoxLDL and carrying human ApoB100 as a transgene (huB100t9) is describedand in particular the hybridoma clone 48-5 deposited according to theBudapest Treaty with the DSMZ-Deutsche Sammlung von Mikro-organismen undZellkulturen GmbH, Inhofftenstrasse 7 B, 38124 Braunschweig, Germany, onJan. 22, 2009 with the accession number DSM ACC2986.

According to a fourteenth aspect, the hybridoma clone 48-5 depositedaccording to the Budapest Treaty with the DSMZ-Deutsche Sammlung vonMikro-organismen und Zellkulturen GmbH, Inhofftenstrasse 7 B, 38124Braunschweig, Germany, on Jan. 22, 2009 with the accession number DSMACC2986 is used to identify a compound inhibiting a CD4⁺ T cell responseto ApoB100. The compound is identified by its capacity to preventactivation of 48-5 upon exposure to apoB100 or a fragment thereof.

The methods and systems herein described can be used in connection withapplications wherein inhibition of CD4+ T cell response to ApoB100,inhibition of CD4 +TRBV31 binding to ApoB100, and/or a therapeutic orpreventive effect for atherosclerosis in an individual is described.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description andexamples sections, serve to explain the principles and implementationsof the disclosure.

FIGS. 1A-1E show diagrams illustrating results related to T cellrecognition of native LDL and ApoB100 according to an embodiment hereindescribed.

FIGS. 2A-2F show diagrams illustrating results supporting an inversecorrelation between oxidation of LDL and T cell activation according toan embodiments herein described.

FIGS. 3A-3D show diagrams illustrating results supporting that in anembodiment herein described hybridoma responses are I-A^(b) restricted.

FIG. 4 shows a diagram illustrating results supporting that in anembodiment herein described hybridoma response is not dependent of CD1and MHC-I presentation.

FIG. 5 shows results of experiments supporting genotyping of the T CellReceptor (TCR) in an embodiment herein described.

FIG. 6 shows diagrams illustrating results related to TCR expressionevaluated by FACS in an embodiment herein described.

FIG. 7 shows a diagram illustrating results indicating plasma levels ofcholesterol and triglycerides according to an embodiment hereindescribed.

FIGS. 8A-8D show diagrams illustrating results indicating antibodytiters to oxLDL and LDL in an embodiment herein described.

FIGS. 9A-9B show a diagram and a photographic representation indicatingthat, in an embodiment herein described, TRBV31 + cell-depletion reducesthe T cell response to ApoB100.

FIGS. 10A-10B show diagrams illustrating results indicating that, in anembodiment herein described immunization against TRBV31 induces blockingantibodies.

FIGS. 11A-11B show diagrams illustrating results indicating that, in anembodiment herein described, immunization against TRBV31 reducesatherosclerosis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Provided herein are methods and systems and related products andcompositions for treating and/or preventing atherosclerosis or acondition associated thereto in individuals.

The term “treating” or “treatment” as used herein indicates any activitythat is part of a medical care for, or that deals with, a conditionmedically or surgically. The term “preventing” or “prevention” as usedherein indicates any activity, which reduces the burden of mortality ormorbidity from a condition in an individual. This takes place atprimary, secondary and tertiary prevention levels, wherein: a) primaryprevention avoids the development of a disease; b) secondary preventionactivities are aimed at early disease treatment, thereby increasingopportunities for interventions to prevent progression of the diseaseand emergence of symptoms; and c) tertiary prevention reduces thenegative impact of an already established disease by restoring functionand reducing disease-related complications.

The term “condition” as used herein indicates as usually the physicalstatus of the body of an individual (as a whole or of one or more of itsparts) that does not conform to a physical status of the individual (asa whole or of one or more of its parts) that is associated with a stateof complete physical, mental and possibly social well-being. Conditionsherein described include but are not limited to disorders and diseaseswherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms. Exemplary conditions include but are not limited to injuries,disabilities, disorders (including mental and physical disorders),syndromes, infections, deviant behaviors of the individual and atypicalvariations of structure and functions of the body of an individual orparts thereof.

The wording “associated to” as used herein with reference to two itemsindicates a relation between the two items such that the occurrence of afirst item is accompanied by the occurrence of the second item, whichincludes but is not limited to a cause-effect relation andsign/symptoms-disease relation.

The term “individuals” as used herein indicates a single biologicalorganism such as higher animals and in particular vertebrates such asmammals and more particularly human beings.

Atherosclerosis is currently viewed as a chronic lipid-related andimmunemediated inflammatory disease of the arterial walls. Many immunecomponents have been identified that participate in atherogenesis andpre-clinical studies have yielded promising results suggesting thatimmunomodulatory therapies targeting these components can reduceatherosclerosis.

The term “atherosclerosis” as used herein indicates a cardiovascularcondition, and in particular a chronic inflammatory diseasecharacterized by the accumulation of lipoproteins eliciting aninflammatory response in the intima of the arterial wall. The tunicaintima (or just intima) is the innermost layer of an artery or vein. Theintima is typically formed by one layer of endothelial cells and issupported by an internal elastic lamina. In the intima the endothelialcells are in direct contact with the blood flow. Adaptive immuneresponses engaging clonally expanded T cell populations contribute tothis inflammatory process, as well as innate immune responses mounted bymacrophages and other cells. Several lines of evidence point tocomponents of the low density lipoprotein (LDL) particles as triggers ofvascular inflammation.

The tee a “Low-density lipoprotein” or “LDL” as used herein indicates atype of lipoprotein that transports cholesterol and triglycerides fromthe liver to peripheral tissues. LDL is one of the five major groups oflipoproteins; these groups include chylomicrons, very low-densitylipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-densitylipoprotein, and high-density lipoprotein (HDL). Like all lipoproteins,LDL enables fats and cholesterol to move within the water based solutionof the blood stream. Typically a native LDL particle contains a singleapolipoprotein B (apoB) molecule that circulates the fatty acids,keeping them soluble in the aqueous environment. The apoB on the LDLparticle acts as a ligand for LDL receptors in various cells throughoutthe body. The protein occurs in the plasma in two main isoforms, ApoB48and ApoB100. The first is synthesized exclusively by the smallintestine, the second by the liver. The Apolipoprotein B-100 moleculehas 4536 amino acid residues and a MW of about 514 kD. Additionally, LDLhas typically a highly-hydrophobic core consisting of a polyunsaturatedfatty acid known as linoleate and about 1500 esterified cholesterolmolecules. This core is surrounded by a shell of phospholipids andunesterified cholesterol as well as a single copy of the ApoB-100. LDLparticles are approximately 22 nm in diameter and have a mass of about 3million Daltons. Low-density lipoprotein receptors sit on the outersurface of many types of cells, where they pick up low-densitylipoproteins circulating in the bloodstream and transport them into thecell. Once inside the cell, the low-density lipoprotein is broken downto release cholesterol. The cholesterol is then used by the cell,stored, or removed from the body. After low-density lipoproteinreceptors drop off their cargo, they are recycled back to the cellsurface to pick up more low-density lipoproteins. When LDL particlesinfiltrate the intima, they are prone to undergo oxidativemodifications. Such changes likely include enzymatic attacks bymyeloperoxidase and lipoxygenases as well as non-enzymatic oxidativereactions. As an initial result of oxidation, double-bonds of fatty acidresidues in phospholipids, cholesterol esters and triglycerides arecleaved, generating reactive aldehydes and truncated lipids. Among thelatter, modified phospholipids such as lysophosphatidylcholine andoxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC)can activate endothelial cells, macrophages and B1-type B cells toinitiate innate immune responses, including adhesion moleculeexpression, chemokine production, and secretion of natural antibodies.The protein moiety of LDL is also a target of oxidative modifications.They include formation of adducts of malondialdehyde (MDA),4-hydroxynonenal and other molecular species on lysyl residues ofapolipoprotein B-100 (ApoB100). Antibodies are formed to MDA-lysine andother oxidatively generated epitopes of LDL particles (Ketelhuth, D. F.,Tonini, G. C., Carvalho, M. D., Ramos, R. F., Boschcov, P., and Gidlund,M. (2008). Autoantibody response to chromatographic fractions fromoxidized LDL in unstable angina patients and healthy controls. Scand JImmunol 68, 456-462). Such antibodies circulate in peripheral blood andcan also be found in atherosclerotic lesions. In contrast to the naturalantibodies to oxidized phospholipids produced by B1 cells,anti-MDA-ApoB100 antibodies are largely IgG molecules (Yla-Herttuala,S., Palinski, W., Butler, S. W., Picard, S., Steinberg, D., and Witztum,J. L. (1994). Rabbit and human atherosclerotic lesions contain IgG thatrecognizes epitopes of oxidized LDL. Arterioscler Thromb 14, 32-40.).This implies the involvement of T cell help to activate isotypeswitching in the B cell.

In several embodiments, the method for treating and/or preventingatherosclerosis in an individual herein described comprises inhibitingin the individual a CD4⁺ T cell response to ApoB100.

The term “T cells” as used herein indicates a group of white blood cellsknown as lymphocytes, which play a central role in cell-mediatedimmunity and can be distinguished from other lymphocyte types, such as Bcells and natural killer cells by the presence of a special receptor ontheir cell surface called T cell receptors (TCR). The abbreviation T, inT cell, stands for thymus, since it is the principal organ in thedevelopment of the T cell. T cells have been identified both inhypercholesterolemic mice and among clones isolated from humanatherosclerotic lesions, the molecular properties of the T cell epitopesare poorly understood due to the biochemical complexity of the LDLparticle and the oxidative process. Antibodies are generally produced byplasma cells, which have matured from B lymphocytes in the presence ofvarious cytokines produced by activated CD4+ T lymphocytes and by directT lymphocyte-B lymphocyte interactions. CD4+ T lymphocytes are activatedwhen they encounter an antigenic peptide with major histocompatibilitycomplex class II antigen-presenting cells, such as B lymphocytes andmacrophages. The recognition of major histocompatibility complex classII peptide complexes by T lymphocytes is therefore central to thedevelopment of immune responses and antibody production. The majorhistocompatibility complex class II molecules are highly polymorphicheterodimeric membrane glycoproteins composed of α and β chains. Thefunction of major histocompatibility complex class II molecules is tobind short peptides derived mainly from extracellular proteins, in turnforming major histocompatibility complex class II peptide complexes thatinteract with appropriate T-cell receptors of CD4+ T lymphocytes. Atmaturity, MHC molecules are anchored in the cell membrane, where theydisplay short polypeptides to T cells, via the T cell receptors (TCRs).All MHC molecules receive polypeptides from inside the cells they arepart of and display them on the cell's exterior surface for recognitionby T cells.

The term “T cell receptor or TCR” indicates a molecule found on thesurface of T lymphocytes (or T cells) that is, in general, responsiblefor recognizing antigens bound to major histocompatibility complex (MHC)molecules. The TCR is a heterodimer consisting of an alpha and betachain in 95% of T cells, whereas 5% of T cells have TCRs consisting ofgamma and delta chains. Engagement of the TCR with antigen and MHCresults in activation of its T lymphocyte through a series ofbiochemical events mediated by associated enzymes, co-receptors, andspecialized accessory molecules. The variable domain of both the TCRa-chain and β-chain have three hypervariable or complementaritydetermining regions (CDRs), whereas the variable region of the 13-chainhas an additional area of hypervariability (HV4) that does not normallycontact antigen and therefore is not considered to be a CDR. CDR3 is themain CDR responsible for recognizing processed antigen, although CDR1 ofthe alpha chain may also interact with the N-terminal part of theantigenic peptide, and CDR1 of the beta chain interacts with theC-terminal part of the peptide. CDR2 regions interact with the MHCmolecule presenting the peptide. The signal from the T cell complex isenhanced by simultaneous binding of the MHC molecules by a specificco-receptor.

The term “CD4⁺ T cells” as used herein indicates T cells, and inparticular helper T cells and regulatory T cells, presenting a coreceptor CD4 on their surface. On helper T cells, the CD4 exclusivelybinds the class II MHC. The co-receptor not only ensures the specificityof the TCR for the correctly-presented antigen but also allows prolongedengagement between the antigen presenting cell and the T cell, thusenhancing the recruitment of essential molecules (e.g. Lck,) inside thecell that are involved in the signaling of that activated T lymphocyte.For example, antigen binding to the T cell receptor (TCR) stimulates thesecretion of IL-2 and several other cytokines, and the expression ofIL-2 receptors (IL-2R).

In some embodiments, the CD4⁺ T cell are CD4+T cell presenting a T cellreceptor beta variable 31 (TCR TRBV31).

The term “present” as used herein with reference to a compound orfunctional group indicates attachment performed to maintain the chemicaland/or biological reactivity of the compound or functional group asattached. Accordingly, protein presented on a cell is able to performunder the appropriate conditions the one or more chemical and/orbiological reactions that chemically and/or biologically characterizethe protein.

The term “T cell receptor beta variable 31” or “TRBV31” as used hereinindicates T cells carrying the T cell receptor beta variable 31, whichis identifiable by a skilled person upon reading of the presentdisclosure.

The term “T cell receptor beta variable 30” or “TRBV30” as used hereinindicates T cells carrying the T cell receptor beta variable 30, whichis identifiable by a skilled person upon reading of the presentdisclosure.

The term “T cell receptor with a DNA sequence highly homologous to thatof TRBV31” as used herein denotes T cell receptors the beta domains ofwhich are orthologs of TRBV31, as defined in the TreeFam database(available online). For example, in humans, the ortholog of TRBV31 isTRBV30.

In some embodiments, a compound inhibiting the CD4+ T cell response toApoB100 is used. Such a compound may be a compound identified by itscapacity to prevent activation of the hybridoma clone 48-5 upon exposureto apoB100 or a fragment thereof. The hybridoma clone 48-5 has beendeposited according to the Budapest Treaty with the DSMZ-DeutscheSammlung von Mikro-organismen and Zellkulturen GmbH, Inhofftenstrasse 7B, 38124 Braunschweig, Germany, on Jan. 22, 2009 with the accessionnumber DSM ACC2986.

In some embodiments, inhibiting the CD4+ T cell response to ApoB100 isperformed by inhibiting the TCR TRBV31.

In some embodiments, inhibiting the CD4+ T cell response to ApoB100 isperformed by inhibiting a T cell receptor with a DNA sequence highlyhomologous to that of TRBV31.

In some embodiments, treatment and/or prevention of atherosclerosisand/or a condition associated thereto in an individual, can be performedby administering a therapeutically effective amount of a compoundinhibiting the binding of the T cell receptor beta variable 31 (TCRTRBV31) to molecules comprising apolipoprotein B-100 or fragmentsthereof.

In some embodiments, treatment and/or prevention of atherosclerosisand/or a condition associated thereto in an individual, can be performedby administering a therapeutically effective amount of a compoundinhibiting the binding of a T cell receptor with a DNA sequence highlyhomologous to that of TRBV31 to molecules comprising apolipoproteinB-100 or fragments thereof.

In some embodiments, the binding of the T cell receptor beta variable 31(TCR TRBV31) to molecules comprising apolipoprotein B-100 or fragmentsthereof is inhibited.

In some embodiments, the binding of a T cell receptor with a DNAsequence highly homologous to that of TRBV31 to molecules comprisingapolipoprotein B-100 or fragments thereof is inhibited.

In some embodiments, inhibition of the CD4+ T cell response to ApoB100is performed by immunizing the individual against TCR TRBV31 or animmunogenic fragment thereof.

Antigen-specific immunomodulation by vaccination is an attractiveapproach to prevent or treat chronic inflammatory diseases. Bymobilizing protective immune responses in an antigen-specific manner,side effects due to hampered host defense against infections areavoided. Therefore, antigen-specific suppression of pathologicautoimmunity is of interest in chronic inflammatory diseases such asatherosclerosis.

Antigen-specific immunoprotection can be achieved through severaldifferent mechanisms, such as production of protective antibodies,deletion or inactivation (anergy) of pathogenic T cell clones, orinduction of suppressive cellular immunity mediated by the family ofregulatory T cells (Treg).

In some embodiments, immunization can be performed with the TCR TRBV31protein. In some embodiments, immunization can be performed with afragment of the TCR TRBV31 and in particular the TRBV31 peptide includespart of the CDR2 variable region of the β chain of the TCR (amino acidresidues 45-62, ATGGTLQQLFYSITVGQV—SEQ ID NO: 1) herein also indicatedas TRBV31 peptide.

The term “protein” or “polypeptide” as used herein indicates an organicpolymer composed of two or more amino acid monomers and/or analogsthereof. The term “polypeptide” includes amino acid polymers of anylength including full length proteins and peptides, as well as analogsand fragments thereof. A polypeptide of three or more amino acids isalso called an oligopeptide. As used herein the term “amino acid”,“amino acidic monomer”, or “amino acid residue” refers to any of thetwenty naturally occurring amino acids including synthetic amino acidswith unnatural side chains and including both D and L optical isomers.The term “amino acid analog” refers to an amino acid in which one ormore individual atoms have been replaced, either with a different atom,isotope, or with a different functional group but is otherwise identicalto its natural amino acid analog.

The term “fragment” as used herein indicates a portion of a polypeptideof any length. An antigenic fragment of TCR TVBR31 is accordingly aportion of TCR TVBR31 that presents antigenic properties. Antigenicfragments of TCR TVBR31 herein described also include any peptideshowever synthesized and possible derivatives thereof.

The term “derivative” as used herein with reference to a firstpolypeptide (e.g., TCR TVBR31 antigenic fragment), indicates a secondpolypeptide that is structurally related to the first polypeptide and isderivable from the first polypeptide by a modification that introduces afeature that is not present in the first polypeptide, while retainingfunctional properties of the first polypeptide. Accordingly, aderivative polypeptide of an antigenic fragment of TCR TVBR31, usuallydiffers from the original polypeptide or portion thereof by modificationof the amino acidic sequence that might or might not be associated withan additional function not present in the original polypeptide orportion thereof. A derivative polypeptide of an antigenic fragment ofTCR TVBR31 retains however antigenic properties comparable to the onesdescribed in connection with TCR TVBR31 or the antigenic fragmentthereof.

In some embodiments, the peptide TCR TRBV31 or a fragment thereof for isdescribed for use as a medicament, and in particular for use in thetreatment of atherosclerosis.

In some embodiments, immunization can be performed with antibodyreactive to the T cell receptor beta variable 31 (TCR TRBV31) or afragment thereof.

In some embodiments, antibody reactive to the T cell receptor betavariable 31(TCR TRBV31) or a fragment thereof are described for use as amedicament, and in particular for use in the treatment ofatherosclerosis.

The term “antibody” as used herein refers to a protein of the kind thatis produced by activated B cells after stimulation by an antigen and canbind specifically to the antigen promoting an immune response inbiological systems. Full antibodies typically consist of four subunitsincluding two heavy chains and two light chains. The term antibodyincludes natural and synthetic antibodies, including but not limited tomonoclonal antibodies, polyclonal antibodies or fragments thereof.Exemplary antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM and thelike. Exemplary fragments include Fab Fv, Fab′ F(ab′)2 and the like. Amonoclonal antibody is an anti-body that specifically binds to and isthereby defined as complementary to a single particular spatial andpolar organization of another biomolecule which is termed an “epitope”.In some forms, monoclonal antibodies can also have the same structure. Apolyclonal antibody refers to a mixture of different monoclonalantibodies. In some forms, polyclonal antibodies can be a mixture ofmonoclonal antibodies where at least two of the monoclonal antibodiesbinding to a different antigenic epitope. The different antigenicepitopes can be on the same target, different targets, or a combination.Antibodies can be prepared by techniques that are well known in the art,such as immunization of a host and collection of sera (polyclonal) or bypreparing continuous hybridoma cell lines and collecting the secretedprotein (monoclonal).

In some embodiments, proteins (including antibodies), peptides and/oragents for inhibiting T cells response herein described are comprised ina composition together with suitable adjuvant and/or excipients.

The term adjuvant as used herein indicates a pharmacological orimmunological agent that modify the effect of other agents (e.g., drugs,vaccines) while having few if any direct effects when given bythemselves. They are often included in vaccines to enhance therecipient's immune response to a supplied antigen while keeping theinjected foreign material at a minimum. Types of adjuvants include:Immunologic adjuvant that stimulate the immune system and increase theresponse to a vaccine, without having any specific antigenic effect initself.

The term excipients as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Exemplaryexcipients can also be used to bulk up formulations that contain verypotent active ingredients, to allow for convenient and accurate dosage.In addition to their use in the single-dosage quantity, excipients canbe used in the manufacturing process to aid in the handling of theactive substance concerned. Depending on the route of administration,and form of medication, different excipients may be used that areidentifiable by a skilled person.

In some embodiments, the compositions comprise (immunogenic) peptidefragments of TCR TRBV31 possibly toxins/toxoids: tetanus toxin,diphtheria toxoid, B subunit of cholera toxin, as well as BSA, HAS,rHSA, KLH, ovalbumin.

In some embodiments, the adjuvants and excipients are pharmaceuticallyacceptable and the resulting composition is a pharmaceuticalcomposition. In some of those embodiments, the pharmaceuticalcomposition is a vaccine.

As disclosed herein, agents for inhibiting the CD4⁺ T cell response toApoB100 and/or binding of TCR TRBV31 with ApoB100, in particular the TCRTRBV31, fragments thereof and/or related antibodies, can be provided asa part of systems to treat and/or prevent. The systems can be providedin the form of arrays or kits of parts.

In a kit of parts, the agents and other reagents to perform an assay todetect the inhibiting and/or immunizing can be comprised in the kitindependently. The agents can be included in one or more compositions,and each agent can be in a composition together with a suitable vehicle.

The terms “detect” or “detection” as used herein indicates thedetermination of the existence, presence or fact of a target in alimited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate. The “detect” or“detection” as used herein can comprise determination of chemical and/orbiological properties of the target, including but not limited toability to interact, and in particular bind, other compounds, ability toactivate another compound and additional properties identifiable by askilled person upon reading of the present disclosure. The detection canbe quantitative or qualitative. A detection is “quantitative” when itrefers, relates to, or involves the measurement of quantity or amount ofthe target or signal (also referred as quantitation), which includes butis not limited to any analysis designed to determine the amounts orproportions of the target or signal. A detection is “qualitative” whenit refers, relates to, or involves identification of a quality or kindof the target or signal in terms of relative abundance to another targetor signal, which is not quantified.

Additional components can include labeled molecules and in particular,labeled polynucleotides, labeled antibodies, labels, microfluidic chip,reference standards, and additional components identifiable by a skilledperson upon reading of the present disclosure. The terms “label” and“labeled molecule” as used herein as a component of a complex ormolecule referring to a molecule capable of detection, including but notlimited to radioactive isotopes, fluorophores, chemiluminescent dyes,chromophores, enzymes, enzymes substrates, enzyme cofactors, enzymeinhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (suchas biotin, avidin, streptavidin or haptens) and the like. The term“fluorophore” refers to a substance or a portion thereof which iscapable of exhibiting fluorescence in a detectable image. As aconsequence, the wording “labeling signal” as used herein indicates thesignal emitted from the label that allows detection of the label,including but not limited to radioactivity, fluorescence,chemiluminescence, production of a compound in outcome of an enzymaticreaction and the like.

In some embodiments, detection of the inhibiting and/or immunizing canbe carried either via fluorescent based readouts, in which the labeledantibody is labeled with fluorophore, which includes, but notexhaustively, small molecular dyes, protein chromophores, quantum dots,and gold nanoparticles. Additional techniques are identifiable by askilled person upon reading of the present disclosure and will not befurther discussed in detail.

In particular, the components of the kit can be provided, with suitableinstructions and other necessary reagents, in order to perform themethods here described. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will, usually be included in the kit. Thekit can also contain, depending on the particular method used, otherpackaged reagents and materials (i.e. wash buffers and the like).

In particular, in some embodiments, disclosed are pharmaceuticalcompositions which contain at least one agent as herein described, incombination with one or more compatible and pharmaceutically acceptablevehicles, and in particular with pharmaceutically acceptable diluents orexcipients. In those pharmaceutical compositions the agent can beadministered as an active ingredient for treatment or prevention of acondition in an individual.

In some embodiments, use of a hybridoma from mice immunized with oxLDLand carrying human ApoB100 as a transgene (huB100t9) is described toidentify suitable agents for inhibition of CD4+ T cell response toApoB100. In particular, the hybridoma clone 48-5 deposited according tothe Budapest Treaty with the DSMZ-Deutsche Sammlung von Mikro-organismenund Zellkulturen GmbH, Inhofftenstrasse 7 B, 38124 Braunschweig,Germany, on Jan. 22, 2009 with the accession number DSM ACC2986 andrelated uses and systems.

Further details concerning the implementation of the hybridomas, agents,compositions, methods herein described including systems for performanceof the methods which can be in the form of kit of parts as well asrelated compositions including agents and other reagents together withsuitable carrier, agent or auxiliary agent of the compositions, andgenerally manufacturing and packaging of the kit, can be identified bythe person skilled in the art upon reading of the present disclosure.

EXAMPLES

The methods and systems herein described are further illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting.

In particular, the following examples illustrate exemplary methods andsystems are based on the inhibition of CD4+ T cell response to ApoB100by immunization performed with a specific peptide from TCR TRBV31. Aperson skilled in the art will appreciate the applicability of thefeatures described in detail for immunization performed with a differentpeptide from TCR TRBV31 or for other methods and systems for inhibitingCD4⁺ T cell response to ApoB100 and in particular CD4⁺ T cell presentingTCR TRBV31 according to the present disclosure.

In the following examples values are expressed as mean±standard error ofthe mean (SEM) unless otherwise indicated. Non-parametric Mann-Whitney Utest was used for pairwise comparisons. Differences between groups wereconsidered significant with p below 0.05.

Example 1 Of Native Human LDL and ApoB100 by T Cell Hybridomas

For these experiments HuB100tg mice (mice carrying human ApoB100 as atransgene) were used to characterize the T cell response to oxLDL. Thesemice express human full-length ApoB100 in the liver as well as gut anddisplay a humanized lipoprotein profile.

Immunization

For the generation of T cell hybridomas 7-week old male human ApoB100transgenic mice, huB100tg (C57BL/6,129-Apobtm2sgy, DNX Transgenics,Princeton, USA) were used.

These mice carry the full-length human APOB gene in which codon 2153 hasbeen changed from a leucine to a glutamine to prevent formation ofApoB48, allowing production of ApoB100 only (Boren, J., Lee, I., Zhu,W., Arnold, K., Taylor, S., and Innerarity, T. L. (1998). Identificationof the low density lipoprotein receptor-binding site in apolipoproteinB100 and the modulation of its binding activity by the carboxyl terminusin familial defective apo-B100. J Clin Invest 101, 1084-1093; Linton, M.F., Farese, R V., Jr., Chiesa, G., Grass, D. S., Chin, P., Hammer, R E.,Hobbs, H. H., and Young, S. G. (1993). Transgenic mice expressing highplasma concentrations of human apolipoprotein B100 and lipoprotein (a).J Clin Invest 92,3029-3037; Yao, Z. M., Blackhart, B. D., Johnson, D.F., Taylor, S. M., Haubold, K. W., and McCarthy, B. J. (1992).Elimination of apolipoprotein B48 formation in rat hepatoma cell linestransfected with mutant human apolipoprotein B cDNA constructs. J BiolChem 267, 1175-1182.).

The mice were first immunized subcutaneously (s.c.) with 50 μg of copperoxidized human LDL (oxLDL) mixed with complete Freund's adjuvant (CFA)and after 2 weeks the mice were boosted with 50 f. 1 g oxLDL mixed withincomplete Freund's adjuvant (IFA). Oxidized human LDL (oxLDL) wasprepared as follows: LDL (d=1.019-1.063 g/mL) was isolated byultracentrifugation from pooled plasma of healthy donors as described byHavel et al. (Havel, R J., Eder, H. A., and Bragdon, J. H. (1955). Thedistribution and chemical composition of ultracentrifugally separatedlipoproteins in human serum. J Clin Invest 34, 1345-1353). Afterisolation, LDL was extensively dialyzed against PBS. 1 mM EDTA was addedto an aliquot of LDL to be used as unmodified LDL. Highly oxidized LDLwas obtained by incubating 1 mL of LDL (1 mg/mL protein content,determined by Bradford, Biorad, USA) in the presence of 20 IJM CUS04 for18 h, at 37° C.

T Cell Hybridoma Generation

After the primary immunization of oxLDL and the booster injection asdescribed above lymph-node (LN) cells were collected and fused withthymoma cells to generate hybridomas as follows:

T cell hybridomas were generated by polyethylene glycol-induced fusionof 5×10⁷ lymph node cells (LN) with 3×10⁷ BW5147 thymoma cells asdescribed by Kappler et al. (Kappler, J. W., Skidmore, B., White, J.,and Marrack, P. (1981). Antigen-inducible, H-2-restricted,interleukin-2-producing T cell hybridomas. Lack of independent antigenand H-2 recognition. J Exp Med 153, 1198-1214). Briefly, LN cells fromthe immunized mice were stimulated with 3 μg/ml oxLDL during 3 daysbefore fusion. After fusion, 1×10⁶ thymocytes were added as feeder cellsand the cell suspensions were plated in 96 well plates and incubated at3rC, 7.5% CO₂. Hypoxanthine-aminopterin-thymidine (HAT) was added to themedium after 24 hours of incubation to select successfully fused cells.Among 268 growing hybridoma cultures, 117 were found to express CD3 andCD4. 23 HAT-resistant monoclonal hybridomas were then cloned by limitingdilution and screened for their reactivity against native LDL, copperoxLDL, and purified unmodified ApoB100.

Screening for Positive Clones

The 23 HAT-resistant monoclonal hybridomas were assessed for activationby their IL-2 production (antigen binding to the T cell receptor (TCR)stimulates the secretion of IL-2) after exposure to the putative antigen(native LDL, copper oxLDL, and purified unmodified ApoB100) in thepresence of syngeneic, irradiated antigen-presenting cells (APC). Suchcells take up and process antigens, leading to presentation of antigenicpeptides bound to MHC molecules. Syngeneic APC, i.e. APC from mice thatcarry the same MHC as the T cells are needed in order to preventactivation of T cells recognizing foreign MHC molecules as antigen. Tcell reactivity was determined in 96 well plate assays with 1×10⁵ Thybridoma cells and 4×1 05 irradiated (1.6 Gy) APCs with the differentantigens. LDL and oxLDL were prepared as discussed in Example 1. ApoB100was obtained as previously described by Wessel et al. (Wessel, D., andFlugge, U. I. (1984) A method for the quantitative recovery of proteinin dilute solution in the presence of detergents and lipids Anal Biochem138, 141-143) with minor modifications. Briefly, to 0.1 ml of LDL (1mg/mL) 0.4 ml of methanol, 0.1 ml of chloroform, and 0.3 ml of waterwere added; the suspension was then mixed vigorously and centrifuged at9000×g for 1 min. The upper phase was removed and 0.3 ml of methanoladded to the lower phase and interphase with precipitated protein, whichwas again vigorously mixed and centrifuged at 9000×g for 2 min to pelletthe protein. In order to obtain soluble and highly pure ApoB100, theprotein pelleted was resuspended in a minimum volume of 10% SDS (Bio-RadLaboratories, Hercules, Calif., USA) solution until completesolubilization. These ApoB100 preparations were then subjected to afirst filtration using a PD-10 column (GE Healthcare, previouslyAmersham Biosciences, Uppsala, Sweden) to remove excess of SDS andsubsequent purification using size-exclusion column Superdex-200 (0.5mL/min, in Tris-HCl pH 7.4). The first peak containing ApoB100 wascollected and the extra peaks containing contaminant protein from theLDL purification procedure were discarded. ApoB100 preparations showedover 90% purity when evaluated in a second injection to Superdex-200column (GE Healthcare, Uppsala, Sweden). Finally, protein concentrationwas determined using Bradford assay (Bio-Rad Laboratories, Hercules,Calif., USA).

APCs were prepared by meshing spleens on nylon filters (1 001-1m)followed by lysis of red blood cells and washing. Concavalin A (ConA)was used as a positive control. Cells were cultured for 24 hours, at3rC, 7.5% CO₂, in Dulbecco's modified Eagle's medium (DMEM) supplementedwith 5% fetal calf serum (FCS). Interleukin 2 (IL-2) was measured byELISA (R&D Systems, Abingdon, United Kingdom) in the supernatant ofcultures and used as a read-out for T cell activation. The results canbe seen in FIG. 1A wherein 1×10⁵ hybridoma cells of each of the twentythree HAT-resistant monoclonal hybridomas were incubated with 4×10⁵irradiated APCs together with 40 μg/mL of LDL, oxLDL, or ApoB100. Mediumwas used as negative control.

Remarkably, from all 23 tested monoclonal T cell hybridomas 11 respondedto native human LDL and ApoB100 but none to oxidized LDL.

TCR Genotyping by Polymerase Chain Reaction (PCR)

The eleven clones that could respond to native LDL and ApoB100 weregenotyped by PCR. Total RNA was prepared from 1×10⁷ hybridoma cells fromeach of the 11 hybridoma clones with RNeasy mini kit (Qiagen, Valencia,Calif., USA) and reversely transcribed into cDNA using Superscript II(Invitrogen, Carlsbad, Calif., USA) with random hexanucleotide primers(pdN6) in the presence of RNasin (Life Technologies, Cergy Pontoise,France). The cDNA produced was amplified using appropriate Vα familyspecific 5′ primers (Table 1) together with a constant-region Cα 3′primer, or relevant Vβ family-specific 5′ primers (Table 2) togetherwith a constant-region Cβ, 3′ primer

TABLE 1 Primers for genotyping of the TRAV SEQ α-chain ID familyPrimer sequence NO  1 TRAV01 5′ TGGATGGTTTGAAGGACAGTG 3′  2  2 TRAV02 5′CTGTTTA TCTCTGCTGACCGG 3′  3  3 TRAV03-3 5′ ACGAAGGACAAGGATTCACTGT 3′  4 4 TRAV04 5′ CTGGAGGACTCAGGCACTTACT 3′  5  5 TRAV06 5′GGTACCCGACTCTTTTCTGGT 3′  6  6 TRAV06D-4 5′ ACCCTTTCAGAAGATGACTTCC 3′  7 7 TRAV06D-5 5′ TTT AAAGTCCCAAAGGCCAA 3′  8  8 TRAV06-6 5′TCCTGAAAGTCA TTACGGCTG 3′  9  9 TRAV06-7 5′ AGAGCCTCAAGGGACAAAGAG 3′ 1010 TRAV07-3 5′ AGACTCCCAGCCCAGTGACT 3′ 11 11 TRAV07-5 5′ACA TCAGAGAGCCGCAACC 3′ 12 12 TRAV080-1 5′ CCCTGCCCAGCT AA TCTT AA T 3′13 13 TRAV09-3 5′ CTGCAGCTGAGATGCAAGTATT 3′ 14 14 TRAV090-1 5′TCCTATGGTGGATCCATTTACC 3′ 15 15 TRAV10 5′ TGGACAGAAAACAGAGCCAA 3′ 16 16TRAV11 5′ CAGGCAAAGGTCTTGTGTCC 3′ 17 17 TRAV12-1 5′ACGCCACTCTCCAT AAGAGCA 3′ 18 18 TRAV13-1 5′ GCTCTTTGCACATTTCCTCC 3′ 1919 TRAV14-1 5′ TGCAGTTATGAGGACAGCACTT 3′ 20 20 TRAV14-3 5′CTGCAGTTATGAGAACAGTGCTT 3′ 21 21 TRA  5′ CCAGACGATTCGGGAAAGTA 3′ 22V15-1/0V6-1 22 TRAV16 5′ TTCCATCGGACTCATCATCAC 3′ 23 23 TRAV17 5′AACCTGAAGAAA TCCCCAGC 3′ 24 24 TRAV19 5′ GGAAGACGGAAGATTCACAGTT 3′ 25 25TRAV20 5′ ACGCTCCT AA TAGACATTCGCT 3′ 26 26 TRAV21 5′GTTCCTCTTCAGGGTCCAGA 3′ 27 27 TRAC 5′ CACCAGCAGGTTCTGGGTTC 3′ 28

TABLE 2 Primers for genotyping of the TRBV TCR  SEQ  β-chain ID familyPrimer sequence NO  1 TRBV01 5′ ACACGGGTCACTGATACGGA 3′ 29  2 TRBV02 5′ATGGACAA TCAGACTGCCTCA 3′ 30  3 TRBV03 5′ TCACTCTGAAAA TCCAACCCA 3′ 31 4 TRBV04 5′ T AAACGAAACAGTTCCAAGGC 3′ 32  5 TRBV05 5′ACGGTGCCCAGTCGTTTTA T 3′ 33  6 TRBV12-1 5′ GGATTCCTACCCAGCAGATTC 3′ 34 7 TRBV12-2 5′ AGA T AAAGGAAACCTGCCCAG 3′ 35  8 TRBV13-1 5′CCAGAACAACGCAAGAAGACT 3′ 36  9 TRBV13-2 5′ GGCT ACCCCCTCTCAGACAT 3′ 3710 TRBV13-3 5′ TGGCTTCCCTTTCTCAGACA 3′ 38 11 TRBV14 5′GCGACACAGCCACCT ATCTC 3′ 39 12 TRBV15 5′ CGCAGCAAGTCTCTTATGGAA 3′ 40 13TRBV16 5′ AT AGATGATTCAGGGA TGCCC 3′ 41 14 TRBV17 5′TGAGAAGTTCCAA TCCAGTCG 3′ 42 15 TRBV19 5′ GAAGGCT ATGATGCGTCTCG 3′ 43 16TRBV20 5′ TTCCCATCAGTCATCCCAAC 3′ 44 17 TRBV21 5′AAAA TGCCCTGCT AAGAAACC 3′ 45 18 TRBV23 5′ CAGCCTGGGAA TCAGAACG 3′ 46 19TRBV24 5′ GCA TCCTGGAAA TCCTATCCT 3′ 47 20 TRBV26 5′AGTGTCCTTCAAACTCACCTT 3′ 48 21 TRBV29 5′ AAAGGATACAGGGTCTCACGG 3′ 49 22TRBV30 5′ GGACAAGTTTCCAA TCAGCCG 3′ 50 23 TRBV31 5′TTCATCCT AAGCACGGAGAAG 3′ 51 24 TRBC1 5′ TGCAA TCTCTGCTTTTGATGGCTC 3′ 52

The nomenclature by the international immunogenetics information system(IMGT) was used for the designation of TCR-V chain usage of T cells. AllTCR-V chain sequences were extracted from the IMGT database availableonline) (Lefranc, M.P., Pommie, C., Ruiz, M., Guidicelli, V., Foulquier,E., Troung, L., Thouvenin-Contet, V., and Lefranc, G. (2003). IMGTunique numbering for immunoglobulin and T cell receptor variable domainsand lg superfamily V-like domains. Dev Comp Immunol 27, 55-77). Forcorrespondence between old and new nomenclatures see the IMGT database.The mastermix for PCR reactions contained 10 mM Tris-HCI, 50 mM KCI, 1.5mM MgCl₂, 2 mM dNTP and 0.2 U/ml Taq polymerase (Invitrogen, Carlsbad,Calif., USA). All primers were added to a final concentration of 0.2 μM.The reactions were carried out for 35 cycles using 94 ° C. (40 sec) fordenaturation, 58 ° C. (40 sec) for annealing and 72 ° C. (1 min) forpolymerization. The PCR products were analyzed on a 1.5% agarose gel andvisualized by gel red staining.

Based on TCR genotyping, three different subgroups were identified fromthe eleven clones that could respond to native LOL and ApoB100, andrepresentative data from each subgroup (15-2, 45-1 and 48-5) are showntogether with a non-responding clone 20 (97-3. As can be seen in FIGS.1B-E there was a clear dose-response for hybridoma clones from eachsubgroup to the unmodified ApoB100 protein. A characterization of thehybridoma clones with regard to TCR type is given in Table 3 below.

In order to make sure that the responsiveness was not dependent on ahumanspecific modification of the protein, LOL and ApoB100 were alsoisolated from huB100^(tg)×Ldlr^(−/−) mice and tested against thehybridomas. These mice produce LDL containing human ApoB100. However,these particles lack posttranslational modifications that occur only inhumans and that could hypothetically elicit immune reactions.

In FIGS. 1B-E 1×10⁵ hybridoma cells were incubated with 4×10⁵ irradiatedAPCs with different concentrations of (□) human ApoB100 purified fromhuman LOL, and (x) transgenic human ApoB100, obtained fromhuB100^(tg)×Ldlr^(−/−) mice. In both experiments, IL-2 secretion wasused as readout of activation. From the graph it is seen that thisrecombinant human ApoB100 was also recognized by the T cell hybridomasubgroups 15-2, 45-1 and 48-5. Therefore, native LDL and human ApoB100contains the T cell epitope(s) recognized by these T cells.

Example 2 T Cells from Mice Immunized with LDL or oxLDL Recognize NativeApoB100

Having previously established that atherosclerotic lesions containoligo-clonal T cells and now finding that hybridomas generated from miceimmunized with oxLDL can recognize native ApoB100 of LDL, it wasquestioned whether such autoimmune responses can occur in polyclonal Tcell populations. This hypothesis was tested by immunization ofhuB100^(tg)×Ldlr^(−/−) mice with LDL or oxLDL (highly oxidized),followed by an in vitro challenge of spleen cells from these mice withoxLDL or native ApoB100. These mice lack the LDL-receptor that isresponsible for eliminating LDL from the circulation. When fed a fattydiet, they develop hypercholesterolemia and atherosclerosis. Immuneresponses to LDL is increased in this disease condition, making itsuitable for analysis of such responses

In-Vitro Proliferation Assay

Spleen cells from huB100^(tg)×Ldlr^(−/−) mice immunized with LDL oroxLDL were isolated and cell suspensions prepared. In 96 well plates,5×10⁵ spleen cells were incubated in duplicate with different antigens,as described below, in 200 μL of serum-free medium, 1:100 BD ITS+ Premix(BD Biosciences, Franklin Lakes, N.J., USA), 1 mg/mL BSA (Sigma-Aldrich,St. Louis, Mo., USA), 10 mmol/L HEPES (Gibco Invitrogen, Carlsbad,Calif., 25 USA), 1 mmol/L Na pyruvate (Gibco Invitrogen, Carlsbad,Calif., USA), 1 mmol/L nonessential amino acids (Sigma-Aldrich, St.Louis, Mo., USA), and 50 μg/mL gentamycin sulfate (Sigma-Aldrich, St.Louis, Mo., USA) for 72 hours, at 37° C. in a humid 5% CO₂ atmosphere.Fifty microliters of H³-Thymidine (Sigma-Aldrich, St. Louis, Mo., USA, 1μCi in serum-free medium) was added and after 18 h of incubation T cellproliferation was evaluated with a scintillation counter (Wallac, Turku,Finland).

In FIG. 2(A) 5×10⁵ spleen cells, from huB100×Ldlr^(−/−) mice s.c.immunized with LDL or oxLDL, were challenged in vitro with 20 μ/mL ofhuman oxLDL or native human ApoB100. Values are expressed as mean±SEM ofthe stimulation index obtained from the H3_Thymidine incorporation.Again, native ApoB100 gave the highest response, whereas oxLDL did nottrigger activation (FIG. 2A). Furthermore, this data shows thatimmunization with either native LDL or oxLDL results in expansion of a Tcell population that recognizes native—but not oxidized—epitopes of theLDL particle.

Example 3 Oxidation of LDL Results in Decreased Recognition of the TCell Epitopes

It was further analyzed the relationship between oxidation andantigenicity of LDL particles by exposing T cell hybridomas to a rangeof LDL particles that had been oxidized by copper for varying lengths oftime, resulting in different degrees of oxidation. Highly oxidized LDLwas obtained by incubating 1 mL of LDL (1 mg/mL protein content,determined by Bradford, Biorad, USA) in the presence of 20 ˜M CUS04 for18 h, at 37° C.; different degrees of oxidation were obtained byincubation of LDL with 20 ˜M CuSO₄ for 1, 2, 4 or 8 hours. The degree ofoxidation was evaluated by TBARS as previously described (Puhl, H.,Waeg, G., and Esterbauer, H. (1994). Methods to determine oxidation oflow-density lipoproteins. Methods Enzymol 233, 425-441) (FIG. 2B).

Spleen cells from huB100tg×Ld/r′-mice immunized with LDL or oxLDL wereisolated and cell suspensions prepared as described in Example 2 above.1.0×10⁵ hybridoma cells from the cell suspensions were incubated for 24hours with 4×10⁵ irradiated APCs together with 40 ˜g/ml of native LDL oroxLDL that had been incubated with 20 ˜M CuSO₄ for 1, 2, 4, 8 or 18hours. After 24 hours incubation IL-2 secretion was evaluated in thesupernatant of cultured cells (FIG. 2C=15-2 clone; FIG. 2D=48-5 clone;FIG. 2E=45-1 clone; FIG. 2F=97-3 negative control clone).

Surprisingly, there was an inverse relationship between the degree ofoxidation and amplitude of activation for all of the T cell hybridomas(FIG. 2C-F). Thus, native LDL gave the highest IL-2 response, whereasheavily oxidized LDL (i.e. LDL that had been oxidized for 18 hours) didnot trigger any activation at all. Effects of oxLDL on cell viabilityafter incubations was tested and showed no significant differencebetween groups (data not shown). These data suggest that the T cellresponse to epitopes in LDL is gradually diminished upon oxidation.

Example 4 T Cell Responses to Native LDL and ApoB100 Depend on SpecificMHC Class II Molecules

Since purified ApoB100 was able to induce activation of the CD4+ T cellhybridomas obtained from the immunized mice, it was hypothesized thatthe epitopes would be peptides presented by the MHC class II complex; inthe case of the present mice, the I-A^(b) haplotype. For evaluation ofMHC class II restriction, 1×10⁵ hybridoma cells were incubated with4×10⁵ irradiated APCs, taken from mice that are either syngeneic donors(C57BL6; I-A^(b)) i.e. I-A^(b) haplotypes, or from mice of a differentgenotype (BALB/c; I-A^(d)), 5 i.e. I-A^(d) haplotypes, together withdifferent concentrations of human ApoB100. In parallel, hybridoma cellswere challenged with ApoB100 in the presence of irradiated APCs of theI-A^(b) haplotype, together with anti-MHC class II blocking antibody.After 24 hours incubation IL-2 secretion was evaluated in thesupernatant of cultured cells. A=15-2 clone; B=48-5 clone; C=45-1 clone;D=97-3 negative control clone.

It can be seen in FIG. 3 that when a blocking antibody against mouseI-A^(b) was added, there was a clear suppression of T cell activationfor all clones. Mismatched I-A^(d) expressing APCs from BALB/c micecould not present ApoB100 to antigen-specific T cell hybridomas.Therefore, recognition of ApoB100 protein by antigen specific T cellsrequires that antigenic protein components are presented by syngeneicMHC class II molecules.

To test if lipid antigens bound to or possibly co-purified with ApoB100were involved, T cell responses to ApoB100 presented by APCs carryingI-A^(b) but lacking CD1d, an MHC-like molecule that presents lipidantigens to T cells, were assessed. However, lack of CD1d on APCs didnot impair the T cell response (FIG. 4). Similarly, APCs from I-A^(b)mice lacking MHC class I molecules were able to present ApoB100 antigensto T cell hybridomas (FIG. 4). These results show that ApoB100 antigenis recognized by MHC class II restricted CD4+ T cells. Together, theexperiments described in this example indicate that cellular immuneresponses to apoB100 are mounted by T cells of the CD4+type and requireantigen presentation involving MHC class II molecules on APC carryingthe same MHC type as the responding T cells. Such a scenario ischaracteristic of classical presentation to T cells of peptide antigenstaken up from the extracellular space.

Example 5 T Cells Reactive to Native LDL and ApoB100 Express TRBV31

The TCR of T cell hybridomas were characterized on the mRNA level usingRT-PCR amplification of rearranged variable domains. RNA isolation andcDNA synthesis was performed as mentioned in Example 1.

The cDNA produced was amplified using appropriate Vβ family-specific 5′primers (Table 2) together with a constant-region Cβ 3′ primer, orrelevant Vα family-specific 5′ primers (Table 1) together with aconstant-region Cα 3′ primer. The design of all primers was based onpreviously published sequences (Lefranc, M. P., Pommie, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., andLefranc, G. (2003). IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains. Dev CompImmunol 27, 55-77). The mastermix for PCR reactions contained 10 mMTris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 1 mM dNTP and 0.2 U/ml Taq polymerase(Invitrogen). All primers were added to a final concentration of 0.2 μM.The reactions were carried out for 35 cycles using 94° C. (40 seconds)for denaturation, 56° C. (40 seconds) for annealing and 72° C. (1minute) for polymerization. The PCR products were analyzed on a 1.5%agarose gel and visualized by ethidium bromide staining.

Real time-PCR was carried out using assay-on-demand primers and probesfor CD3 and hypoxanthine guanidine ribonucleosyl transferase (HPRT)(Applied Biosystems, Foster City, Calif., USA) in an ABI 7700 SequenceDetector (Applied Biosystems, Foster City, Calif., USA).

For quantitative TRBV31 expression analysis, genotyping primers wereused in combination with a probe that was designed based on thenucleotide sequences of the constant region of TCR β chain(5′-TCCACCCAAGGTCT-3′-SEQ ID NO:53). The probe was designed using ABIPrimer Express software (Applied Biosystems, Foster City, Calif., USA)and it was synthesized with a 6-carboxy-fluorescein (FAM) reportermolecule attached at the 5′ end (Applied Biosystems, Foster City,Calif., USA). Data was analyzed on the basis of the relative expressionmethod with the formula 2-ΔΔCT, where ΔΔCT=ΔCT (sample)−ΔCT(calibrator=average CT values of all samples within each group), and ΔCTis the CT of the housekeeping gene (HPRT) subtracted from the CT of thetarget gene (Giulietti, A, Overbergh, L., Valckx, D., Decallonne, B.,Bouillon, R., and Mathieu, C. (2001). An overview of real-timequantitative PCR: applications to quantify cytokine gene expression.Methods (San Diego, Calif. 25, 386-401). The results are summarized inTable 3.

TABLE 3 PCR Flow cytometry Resulting Vα gene Vβ gene Anti-TRAV14Anti-TRAV12 Anti-TRAV31 phenotype 15-2 TRAV14, 3 and 20 TRAV31 and 12.1negative — Positive TRAV3/TRBV31 45-1 TRAV4 and 20 TRAV31 and 12.1 — —Positive TRAV4/TRBV31 48-5 TRAV12, 13 and 20 TRAV31 and 12.1 — NegativePositive TRAV13/TRBV31 

The fusion-partner thymoma BW5147 used to generate the hybridoma cells(see Example 1) expressed the rearranged TRAV20 and TRBV12.1 variablechains and all hybridomas were also expressing these chains at the mRNAlevel (Tables 1, 2 and 3 and FIG. 5). All T cell hybridomas specific fornative human LDL and ApoB 100 uniformly expressed the TCR TRBV31 (Tcells carrying the T cell receptor beta variable 31) and no other Vβfamily was identified among them. In contrast, the usage of Vα chainsamong the reactive hybridomas included different families; TRAV3, 4 and13 for 15-2, 45-1 and 48-5 respectively (Table 3). For the non-reactivehybridomas, Vβ as well as Vα TCR variable chains were expressed in anon-restricted fashion, and did not include TRBV31 (data not shown). Ineach one of the LDL-responsive hybridomas, surface expression of theTRBV31 T cell receptor chain was confirmed by flow cytometry analysis(Table 3 and FIG. 6). All commercially available anti-mouse TCR-Vα andTCR-Vβ monoclonal antibodies (mAb, BD PharMingen, San Diego, Calif.,USA) were used to stain the TCR-Vα and TCR-Vβ on the selected T cellhybridomas. The TCR-V mAbs were conjugated to PE, FITC orbiotin/streptavidin-Cy5. In combination with these antibodies,anti-CD3-Pacific Blue and anti-CD4-APC were used. Spleen cells fromnon-immunized mice were used as positive controls for all antibodies.The cells were analyzed on a CyAn™ ADP flow cytometer (Dako, Glostrup,Denmark).

Example 6 Depletion of TRBV31⁺ T Cells Reduces the ProliferativeResponse to ApoB100

To directly test the overall importance of the TRBV31 variable chain forthe recognition of ApoB100, mice were immunized and boosted with ApoB100followed by in vitro depletion of TRBV31+T cells from spleen.HuB100^(tg)×Ldlr^(−/−) mice were immunized and boosted s.c. withApoB100. Spleen cells were harvested and followed by in vitro depletionof TRBV31+ or TRBV19+ T cells from spleen by Fluorescence-Activated CellSorting (FACS). 60×10⁶ spleen cells were split in 2 and stainedseparately with anti-TRBV31 and anti-TRBV19 (PharMingen, San Diego,Calif., USA) respectively. TRBV19 was used as a control for the sortingprocedure since none of the hybridomas recognizing ApoB100 presentedTRBV19 usage. Of note, the clones 96.7 and 97.3, expressing theTRBV191TRAV13.2 chains with no recognition of LDL or ApoB100, did showreactivity to HDL (data not shown). After staining the cells they weresorted in a MoFlo Cytomation cell sorter (Cytomation Bioinstruments,Freiburg, Germany) for the depletion of positive events. Negative cellswere then collected and used in the proliferation assay against ApoB100.

Thereafter 5×10⁵ TRBV31+/TRBV19− or TRBV19+/TRBV31− spleen cells werechallenged in vitro with different concentrations of human ApoB100.Stimulation index was obtained by H3-thymidine incorporation asdescribed in Example 2. The depletion of TRBV31+ T cells from spleen ledto a significant reduction in the response to the ApoB100 antigen uponin vitro challenge, which was not observed when T cells expressing thevariable chain, TRBV19, were depleted from the spleenocyte population(FIG. 7). Therefore, a significant proportion of the cellular immuneresponse to ApoB100 in this model is carried out by TRBV31+ T cells.

Example 7 Immunization Against TRBV31+TCR Protects AgainstAtherosclerosis

To determine the impact on atherosclerosis of the ApoB100-reactiveTRBV31+ T cell population, huB100^(tg)×Ldlr^(−/−) mice were immunizedwith a peptide derived from TCR TRBV31. In these experiments, elevenweek-old male huB100^(tg)×Ldlr^(−/−) mice (C57BL/6,129-Apob^(tm2Sgy)Ldlr^(tm1Her) (Skalen, K., Gustafsson, M., Rydberg, E.K., Hulten, L. M., Wiklund, O., Innerarity, T. L., and Boren, J. (2002).Subendothelial retention of atherogenic lipoproteins in earlyatherosclerosis. Nature 417, 750-754) kindly provided by Jan Boren,Göteborg University) were used. These mice have an elevated serum 30cholesterol level of 200-400 mg/dl and they have very high levels(>2,000 mg/dl) when fed a high fat diet. The mice were immunized s.c.with 100 μg of TRBV31 peptide This TRBV31 peptide includes part of theCDR2 variable region of the β chain of the TCR (amino acid residues45-62, “ATGGTLQQLFYSITVGQV”—SEQ ID NO:1). The peptide is synthesized andconjugated to keyhole limpet hemocyanin (KLH) by Anaspec, San Jose,Calif., USA).

KLH is a natural protein isolated from the marine mollusk keyhole limpetand is an immunogenic carrier protein that, in vivo, increases antigenicimmune responses to haptens and other weak antigens such as idiotypeproteins. The TRBV31 peptide-KLH conjugate was emulsified with completeFreund's adjuvant, and the mice were boosted 4 weeks later with the sameantigen emulsified with IFA. A control group of mice was immunized s.c.with 100 μg of KLH using the same protocol as for the peptide. The micewere kept on high fat diet (0.15% cholesterol) starting 5 days after theimmunization until sacrifice 10 weeks later with CO₂. In addition,irradiated spleen cells from mice on C57BL16 background were used asantigen presenting cell (APC) in the hybridoma experiments. Allexperiments were approved by the local ethics committee.

Subcutaneous immunization with this TRBV31 peptide, representing part ofthe CDR2 domain and fused to a carrier protein, in Freund's adjuvantinduced the production of antibodies to TCR TRBV31 (FIGS. 8A-D). Thetiters of specific antibodies to TRBV31 were measured by ELISA. Briefly,50 μL of the TRBV31 peptide (5 μg/mL in PBS pH 7.4) were added to 96well ELISA plates and incubated overnight at 4° C. Coated plates werewashed with PBS and thereafter blocked with 1% Gelatin (GibcoInvitrogen, Carlsbad, Calif., USA) in PBS for 1 hour, at roomtemperature. Next, plates were washed and followed by 2 more hours ofincubation with mouse plasma diluted in Tris buffered saline(TBS)/gelatine 0.1%. After washing, total IgG, and igG isotypes weredetected using enzyme-conjugated anti-mouse antibodies (BD Biosciences,Franklin Lakes, N.J., USA). The plates were washed and the colorreaction developed using TMB substrate reagent (BD Biosciences, FranklinLakes, N.J., USA). The absorbance was measured using a microplate reader(VersaMax, Molecular Devices, Sunnyvale, Calif., USA). FIG. 8A shows IgGantibody titers to TRBV31 peptide measured by ELISA. This datademonstrates that immunization induced high-titer antibodies to thepeptide dervived from TRBV31.

Affinity purified circulating IgG antibodies from immunized mice stainedLDL-reactive TRBV31+hybridomas (48-5 clone) (FIG. 8B), indicating thatthese IgG antibodies bound to TCR of such cells. Total IgG plasmaantibodies from KLH or TRBV31 immunized mice were affinity purifiedusing a protein-G column (GE healthcare, Uppsala, Sweden). 1×10⁴hybridoma cells (clone 48-5) were cultured with 40 μ/mL of ApoB100 inthe presence of 4×10⁴ irradiated APCs. For the blocking of hybridomaactivation, the different antibodies (KLH IgG or TRBV31 IgG) were addedat the beginning of culture, at the concentrations 0.1, 1, 10 and 100μg/ml, as indicated in FIG. 8C and were present throughout. After 24 hof incubation, IL-2 was measured in the supernatant. In FIG. 8C it canbe seen that IgG from TRBV31-peptide immunized mice inhibited activationof T cell hybridoma (clone 48-5) in response to ApoB100. Thus,immunization led to production of antibodies that prevented TCR TRBV31recognition of antigen. The Hybridoma clone 48-5 has been depositedaccording to the Budapest Treaty with the DSMZ-Deutsche Sammlung vonMikro-organismen und zellkulturen GmbH, Inhoffenstraβe 7 B, 38124Braunschweig, Germany, on Jan. 22, 2009 with the accession number DSMACC2986.

Finally, the role of TRBV31⁺ T cells in atherosclerosis was tested inexperiments with HuB100^(tg)×Ldlr^(−/−) mice. These animals wereimmunized with the TRBV31-peptide conjugated to KLH carrier protein,followed by a single booster injection; atherosclerotic lesions wereanalyzed 10 weeks after immunization. Mice that were immunized with KLHonly were used as control.

After sacrifice, blood from mice was collected through cardiac puncture.This was followed by vascular perfusion with sterile RNase-free PBS.Thoracic aorta and heart were dissected and saved for lesion analysis.Two thirds of the spleen was saved for cell experiments and one thirdsnap-frozen for later RNA isolation. Draining lymph nodes from theinguinal region and also the abdominal aorta were snap-frozen and savedfor RNA isolation. Lesion analysis was performed as described previously(Nicoletti, A., Kaveri, S., Caligiuri, G., Bariety, J., and Hansson, G.K. (1998). Immunoglobulin treatment reduces atherosclerosis in apo Eknockout mice. J Clin Invest 102, 910-918). Briefly, hearts weresubjected to serial cryostat sections from the proximal 1 mm of theaortic root. Hematoxylin/oil red O-stained sections were used for lesionsize evaluation using Image J software (NIH, Bethesda, Md., USA). InFIG. 9A the mean lesion size was determined after measuring 8 sectionscollected at every 100 I-lm over a 1-mm segment of the aortic root. FIG.9B shows images captured for each section and the surface areas of thelesion(s) and of the entire vessel were measured. The fraction arealesion (%) is the ratio between the cross-section area occupied bylesion and the total vessel cross-section area.

It can be seen that immunization with the TRBV31-peptide led to adramatic and highly significant 70% reduction of lesion size in theaortic root as compared to control mice immunized with KLH carrierprotein alone (P<0.01).

This effect was paralleled by induction of anti-TRBV31 antibodies butdid not affect antibody titers to oxLDL, plasma cholesterol ortriglyceride levels (FIGS. 10 and 11).

Plasma cholesterol and triglycerides were evaluated by enzymaticcolorimetric specific kits (Randox Lab. Ltd. Crumin, UK) according tothe manufacturer's protocol.

TCR TRBV31 mRNA was quantified by real time-PCR in aorta of bothimmunized groups as follows: RNA isolation and cDNA synthesis wasperformed as previously mentioned (see above). Real time-PCR was carriedout using assay-on-demand primers and probes for CD3 and hypoxanthineguanidine ribonucleosyl transferase (HPRT) (Applied Biosystems, FosterCity, Calif., USA) in an ABI 7700 Sequence Detector (Applied Biosystems,Foster City, Calif., USA). For TRBV31 expression analysis, thegenotyping primers were used in combination with a probe that wasdesigned based on the nucleotide sequences of the constant region of TCR13 chain (5′-TCCACCCAAGGTCT-3—SEQ ID NO:54). The probe was designedusing ABI Primer Express software (Applied Biosystems, Foster City,Calif., USA) and it was synthesized with a 6-carboxy-fluorescein (FAM)reporter molecule attached at the 5′ end (Applied Biosystems, FosterCity, Calif., USA). Data was analyzed on the basis of the relativeexpression method with the formula 2ΔΔCT, where ΔΔCT=ΔCT (sample)−ΔCT(calibrator=average CT values of all samples within each group), and ΔCTis the CT of the housekeeping gene (HPRT) subtracted from the CT of thetarget gene (Giulietti et al., 2001).

TCR TRBV31 in RNA was present in the aorta of immunized as well ascontrol mice (FIG. 10D), confirming that this population of T cells wasnot eliminated but prevented from recognizing their cognate antigen. Inconclusion, abrogation of TCR TRBV31 recognition of native ApoB100protein inhibits atherosclerosis.

It is evident that oxLDL particles trigger a strong immune response (forexample, see Palinski, W., Yla-Herttuala, S., Rosenfeld, M. E., Butler,S. W., Socher, S. A., Parthasarathy, S., Curtiss, L. K., and Witztum, J.L. (1990). Antisera and monoclonal antibodies specific for epitopesgenerated during oxidative modification of low density lipoprotein.Arteriosclerosis 10, 325-335 and Zhou X et al, Arterioscler Thromb VascBiol 21: 108-114, 2001). Since T-cell dependent antibodies are formed toaldehyde adducts on ApoB100 and exposure of APC-T cell cultures to oxLDLcan elicit CD4+ T cell activation, it has been assumed that T cellsrecognize epitopes on ApoB100 induced by oxidation of the nativeapolipoprotein. Instead, the present invention shows that T cells fromoxLDL immunized mice preferentially recognize motifs on native LDL.These epitopes are components of the native ApoB100 protein and theirimmunoreactivity is extinguished rather than increased by oxidativemodification of the LDL particle.

The cellular immune response to LDL identified in the present study wasmounted by CD4+ T cells and exhibited MHC class II restriction. This andthe fact that purified ApoB100 protein elicited an identical response asthe intact LDL particle strongly suggest that intracellular processingof ApoB100 in the antigen-presenting cell generated oligopeptideepitopes that were recognized by the T cells as peptide-MHC complexes.The fact that I-Ab was required for the T cell response and could not besubstituted by another MHC class II molecule, I-Ad, further supports thenotion that specific oligopeptides bound to MHC constitute the ligandwith which clonotypic TCR could interact.

Since APC have such a high capacity to present ApoB100 epitopes, thereis a significant risk for autoimmune reactions to LDL. Systemicreactions of this kind could obviously be detrimental since LDL ispresent throughout the circulatory system and in all organs. It waspreviously assumed that all ApoB100 reactive T cell clones were deletedin the thymus during early life, i.e. autoimmunity is avoided by centraltolerance. The current data rule out this possibility. It was clearlydemonstrated that LDL reactive T cells were present inhuApoB100^(tg)×Ldlr^(−/−) mice. In line with these findings, it has beensuggested that the immune system may not be tolerized at all toward manyperipheral antigens, and that the existence of autoreactive T cells perse may not pose an autoimmune danger in the healthy individual.Consequently, ApoB1 OO-reactive T lymphocytes are most likely part ofthe peripheral cell repertoire.

If autoreactivity is not completely eliminated by central tolerance inearly life, autoimmune reactions must be avoided by peripheral tolerancemechanisms. They depend on active inhibition of autoreactivity, e.g. bycells secreting immunoregulatory cytokines such as M2 macrophages andregulatory T cells. Furthermore, proteins synthesized in the liver havebeen reported to preferentially induce tolerogenic immunity. SinceApoB100 is produced in the liver, it may escape autoimmune attack undernormal circumstances. However, accumulation in the artery wall underconditions that favor activation of Th1 effector cells may lead to breakof tolerance and induction of immune reactions to ApoB100 components.

The present data pinpoint CD4+ T cells carrying TCR TRBV31 andrecognizing native ApoB100 protein of LDL as proatherogenic contributorsto the disease process. However, they do not rule out the involvement ofother antigens and immune cells. Thus, it cannot be ruled out thatcertain types of LDL modifications induce autoimmune reactions towardsthe particle. The oxidative changes induced in the particle in vivo maydiffer from those induced by metal ions such as copper. Furthermore, thehybridoma strategy provides detailed information on a small subset ofcells and certain reactivities may not have been represented in thehybridoma repertoire analyzed. Finally, the present strategy focused onantigens presented by professional APC through the endocytic, MHC classII restricted pathway to CD4+ T cells. Additional importantcontributions to LDL immunoreactivity may arise from NKT cellsrecognizing lipid antigens presented via CD1, CD8+ T cells recognizingMHC class I restricted antigens and B cells.

Immunization of atherosclerosis-prone huApoB100×Ldlr^(−/−) mice againstTCR TRBV31 provided important insights into the immunopathology ofatherosclerosis. Antibodies isolated from hyperimmune sera blockedactivation of T cells in response to ApoB100, and elimination of TCRTRBV31+ cells by flow cytometry blunted T cell responses to ApoB100 inspleen cell cultures. However, antibodies induced by the presentimmunization strategy blocked antigen recognition by TCR TRBV31+cellsbut did not eliminate them from aorta.

The induction of blocking anti-TRBV31 antibodies was associated with a70% reduction of atherosclerosis in huApoB100^(tg)×Ldlr^(−/−) mice. Themagnitude of reduction is even better than that achieved by immunizationwith LDL preparations in similar models Urban, R. G., Chicz, R. M., andStrominger, J. L. (1994). Selective release of some invariantchain-derived peptides from HLA-DR 1 molecules at endosomal pH. J ExpMed 180, 751-755; Freigang, S., Horkko, S., Miller, E., Witztum, J. L.,and Palinski, W. (1998). Immunization of LDL receptor-deficient micewith homologous malondialdehyde-modified and native LDL reducesprogression of atherosclerosis by mechanisms other than induction ofhigh titers of antibodies to oxidative neoepitopes. Arterioscler ThrombVase Biol 18, 1972-1982) and therefore strongly suggests that a subsetof T cells recognizing ApoB100 epitopes play a major role in thedevelopment of atherosclerosis. Since TCR TRBV31 did not disappear fromthe aorta after immunization, blocking their recognition of MHC-antigencomplexes may suffice to inhibit the disease process.

The use of the huApoB100^(tg)×Ldlr^(−/−) model permitted the use of welldefined human LDL preparations for dissecting the cellular autoimmuneresponse in atherosclerosis.

According to the above examples T cell recognition of oxLDL, T cellhybridomas from mice immunized with oxLDL were created, which mice werecarrying human ApoB100 as a transgene (huB100^(tg)). These mice producehigh levels of ApoB100 and are also expected to be tolerant to nativehuman LDL.

Hence the experiments exemplified in the above examples show that togreat surprise, T cell responses against native LDL and purified ApoB100in such mice carrying T cell hybridomas from mice immunized with oxLDLand carrying human ApoB100 as a transgene (huB100t9) were registered,whereas oxidation of LDL blunted these responses. The responding T cellswere MHC class II restricted CD4+ cells and expressed a T cell receptor(TCR) containing a variable 13 domain of the TRBV31 type. Elimination ofTRBV31+ T cells attenuated the cellular immune response to ApoB100, andimmunization of hypercholesterolemic mice against a peptide derived fromTCR TRBV31 inhibited the development of atherosclerosis.

These results strongly suggest that autoimmune T cells recognizingprotein epitopes from native LDL promote atherosclerosis.

In summary, in several embodiments, T cell responses against modifiedLDL were investigated by immunizing mice with oxLDL. T cell hybridomaswere established from such mice and analyzed for their reactivitytowards oxidized and native forms of LDL. None of the reactive clonesresponded to oxidized LDL but only to native LDL and purifiedapolipoprotein B-100. Responding hybridomas were CD3+4+8−, restricted byMHC class II antigen I-Ab, and expressed one single T cell receptorvariable (V) beta chain (TRBV31) in combination with different V alphachains. Immunization of huApoB100^(tg)×Ldlr^(−/−) mice against TRBV31reduced atherosclerosis, in parallel with the development of anti-TRBV3antibodies that blocked T cell recognition of ApoB100.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, peptides, proteins, methodsand systems of the disclosure, and are not intended to limit the scopeof what the inventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the disclosure pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference.

It is to be understood that the disclosures are not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular fowls “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. The term “plurality”includes two or more referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of theproducts, methods and system of the present disclosure, exemplaryappropriate materials and methods are described herein as examples andfor guidance purpose.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

The invention claimed is:
 1. A method to treat atherosclerosis in anindividual, the method comprising: administering a therapeuticallyeffective amount of a compound inhibiting in the individual a CD4⁺ Tcell response to ApoB100, wherein the compound comprises T cell receptorbeta variable 31 (TCR TRBV31) or T cell receptor beta variable 30 (TCRTRBV30), or an immunogenic fragment thereof; or an antibody reactive toTRBV31 or TRBV30 or an immunogenic fragment thereof; and whereinadministration of the compound treats atherosclerosis in the individual.2. The method of claim 1, wherein the compound inhibiting the CD4⁺ Tcell response to ApoB100 is a compound that has been identified to havea capacity to prevent activation of a hybridoma clone 48-5 upon exposureto ApoB100 or a fragment thereof, wherein the hybridoma clone 48-5 isthe hybridoma clone that has been deposited according to the BudapestTreaty with the DSMZ-Deutsche Sammlung von Mikro-organismen andZellkulturen GmbH, lnhofftenstrasse 7 B, 38124 Braunschweig, Germany, onJan. 22, 2009 with the accession number DSM ACC2986.
 3. The method ofclaim 1 wherein the CD4⁺ T cell is a CD4⁺ T cell presenting a T cellreceptor beta variable 31 (TCR TRBV31).
 4. The method of claim 1,wherein the CD4⁺ T cell is a T helper cell.
 5. The method of claim 1,wherein the inhibiting is performed by inhibiting the binding of TRBV31to ApoB100 or a fragment thereof.
 6. A method to treat atherosclerosisin an individual, the method comprising: immunizing the individualagainst T cell receptor beta variable 31 (TRBV31) or T cell receptorbeta variable 30 (TRBV30), wherein the immunizing is performed byadministering T cell receptor beta variable 31 (TCR TRBV31) or T cellreceptor beta variable 30 (TCR TRBV30) or an immunogenic fragmentthereof, and wherein the administration treats atherosclerosis in theindividual.
 7. The method of claim 6, wherein the immunizing isperformed by administering an immunogenic fragment of CDR2 variableregion of TCR TRBV31.
 8. The method of claim 6, wherein the immunizingis performed by administering a peptide having the sequence of SEQ IDNO:1 or an immunogenic fragment thereof.
 9. The method of claim 6,wherein the immunizing is performed by administering an immunogenicfragment of CDR2 variable region of TCR TRBV30.